DESCRIPTION: The goal of this proposal is to further characterize the function of Req protein and define its putative role as a transcriptional regulator in the programmed cell death pathway of myeloid cells. In Aim 1, the subcellular localization, phosphorylation state, and physical interaction of Req with dsDNA or other cellular proteins will be determined. The effect of IL-3 receptor occupancy on these properties of Req will also be examined. In Aim 2, experiments will be performed to determine if Req is a sequence- specific DNA binding protein and it functions as an activator of the basal transcription apparatus and/or a repressor of activated transcription. Sequence-specific DNA recognition will be determined by repetitive cycles of selection (Req binding) and PCR amplification of oligonucleotides from a 10 bp randomly-generated combinatorial library. A method that will use 200-300 bp genomic DNA fragments as the Req target sequence will be used to confirm and extend the DNA recognition sequence and to obtain flanking genomic DNA sequences of potential Req-responsive genes. Transcriptional activation/repression will be measured by cotransfection of a req mammalian expression plasmid with a reporter gene construct containing repeats of the cis- acting DNA consensus sequence for Req. More detailed analysis of potential activation or repression domains will be accomplished using chimeric fusion proteins between Req and the DNA binding domain of GAL4. In Aim 3, putative Req primary response genes will be identified.